Natural polycationic antimicrobial peptides have been found in many different species of animals and insects and shown to have broad antimicrobial activity. In mammals, these antimicrobial peptides are represented by two families, the defensins and the cathelicidins. Nearly all of these peptides have membrane affinity, and can permeate and permeabilize bacterial membranes, resulting in injury, lysis, and/or death to the microbes. In particular, the human peptides known as defensins are produced by mammalian and avian leukocytes (e.g. neutrophils, some macrophages) and epithelial cells.
Three defensin subfamilies exist in vertebrates: alpha-defensins, beta-defensins, and circular (theta) minidefensins. All derive from an ancestral gene that existed before reptiles and birds diverged, contain six cysteines, and have largely beta-sheet structures that are stabilized by three intramolecular disulfide bonds. RTD-1, a theta minidefensin, was recently detected in bone marrow from the rhesus monkey, Macacca mulatta. It had 18 residues and was circular, having been formed by the fusion of two truncated alpha-defensin precursors (“demidefensins”) each of which contributed 3 cysteines to the mature peptide. It is not yet known if the cellular machinery responsible for processing these precursors remains operational in human leukocytes.
Alpha-defensins are largely beta sheet peptides that contain 29-35 amino acid residues, including 6 cysteines that form three intramolecular disulfide bonds. Because of the nature of the cysteine pairings, the molecules are effectively macrocyclic. Four of these α-defensins, HNP 1-4, occur primarily in human neutrophils. HD-5 & 6 are found in Paneth cells, specialized cells of the small intestine's crypts. Human α-defensin genes contain three exons and two introns and are clustered on chromosome 8p23. They encode preprodefensins that contain ˜100 residues which include a signal peptide, a polyanionic propiece and the C-terminal defensin domain. Mature defensins are processed by sequential proteolysis.
Beta defensins are generally larger than α-defensins (35-40 residues) and may also be more ancient, since they occur in birds as well as mammals. Beta defensins are expressed in many different types of epithelial cells, and in some glands. In some cases, expression is constitutive; in others, it is inducible. Several β-defensin genes are located on 8p23, adjacent to the α-defensin genes-consistent with their common evolutionary ancestry. The disulfide pairing motif of beta defensins differs from that of α-defensins, however α and β-defensins have generally similar shapes.
The three-dimensional structure of many defensins comprises a complexly folded amphiphilic beta-sheet, with the polar face formed by its arginines and by the N- and C-terminal residues playing an important role in defining microbicidal potency and the antimicrobial spectrum. The antimicrobial effects of defensins are derived from their ability to permeabilize cell membranes and interact with viral envelopes, thereby exposing contents of the microorganism to the environment or abrogating viral infectivity. (See Gudmundsson et al. (1999) J Immunol Methods 232(1-2):45-54.) Antimicrobial peptides are reviewed by Hancock and Lehrer (1998) Trends in Biotechnology 16:82.
In general, the antiviral activities of antimicrobial peptides have not been extensively investigated. Although studies have reported that antimicrobial peptides, such as human neutrophil-derived defensins (α-defensins), are directly virucidal against herpes simplex virus (HSV), and adenovirus strains, only a few reports deal with anti-HIV-1 activity. T22 and T140, analogs of polyphemusins (peptides from horseshoe crabs), are active in inhibiting HIV-1 replication through binding to the chemokine receptor CXCR4. However, these peptides only inhibit the T cell-tropic (T-tropic; X4) strains that utilize CXCR4 as a coreceptor for entry and they are ineffective against strains that utilize CCR5 for entry (macrophage (M)-tropic “R5” viruses). Since sexual transmission is largely attributed to R5 infection, the potential of T22 and T140 as topical vaginal or rectal microbicides is limited.
One study indicated that protegrins (porcine-derived peptides) can inactivate HIV-1 virions. Another study showed that indolicidin, a 13 amino acid peptide isolated from bovine neutrophils, was reproducibly virucidal against HIV-1 only at very high concentrations (333 μg/ml) of peptide. Certain structural and functional similarities exist between the loop motifs of α-defensins and peptides derived from HIV-1 gp41 that may be required for viral fusion and infectivity.
Vaginal and rectal subepithelial stromal tissues are densely populated with dendritic cells (DC), macrophages and T-cells that express both CD4 and the HIV-1 coreceptors, CXCR4 and CCR5. Mechanisms whereby HIV-1 journeys across the mucosal epithelia are not clear, but may directly involve the epithelial cells. Once the virus reaches the lamina propria, it can either directly infect macrophages or T-cells or adhere to or infect DC whose traffic to the regional lymph nodes conveys them into sites of vigorous viral replication. A recent report suggests that binding of HIV-1 to DC is mediated by the C-type lectin DC-SIGN, independent of CD4 or chemokine receptors. Thus, mucosal factors which modulate steps in this process could affect the probability of transmission of HIV-1 infection.
There is a clinical need for novel antiviral and antimicrobial agents that have low toxicity against mammalian cells. The present invention addresses this need.
Relevant Literature
Defensins are reviewed by Lehrer et al. (1992) Ann. Rev. Immunol. 11:105-128. Other endogenous antimicrobials are reviewed in Schonwetter et al. (1995) Science 267:1645-1648; Schroder (1999) Cell Mol Life Sci. 56:32-46 (1999); and Harwig et al. (1994) FEBS Lett 342:281-285.
Specific defensins are described in Tang et al. (1999) Science 286:498-502; Zimmermann et al. (1995) Biochemistry 34:13663-13671; Liu et al. (1997) Genomics 43:316-320; and Palfree & Shen (1994) GenBank U10267; Polley et al GenBank AF238378 disclose the sequence of Homo sapiens chromosome 8p23 clone SCb-561b17.
Retrovirus infection and antiretroviral therapy are discussed in Wilson et al. (1995) J. Infect. Dis. 172:88-96; Wong et al. Science 278:1291-1295; and Yang et al. (1999) J. Virol. 73, 4582-4589.